The EpiTect MSP Kit includes the novel genetically engineered HotStarTaq d-Tect Polymerase in a convenient master mix solution. Higher specificity of this polymerase produces reliable results. The polymerase efficiently discriminates between single-base mismatches at the 3' end of primers during annealing and extension. Since a single mismatch is sufficient for specific MSP results, the EpiTect MSP Kit even enables reliable MSP analysis of single CpG sites.

The EpiTect MSP Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Single base discrimination.

[A] Both primers used for methylated LDOC1 PCR were modified such that a mismatch was introduced at the 3' end of either the forward or the reverse primer. This modification mimics the situation of unchanged primers annealing to bisulfite converted DNA, which differs due to a missing methylation at one CpG site. [B] Using HotStarTaq d-Tect Polymerase, PCR was only successful in the absence of a mismatch at the 3' end of the primer used. A single mismatch is sufficient for correct MSP results.

Sensitivity of HotStarTaq d-Tect.

PCR for methylated [A] p16, [B] NEFH, and [C] NPTX2 were performed using different amounts of methylated DNA (0 pg, 100 pg, and 1 ng) against a background of 10 ng unmethylated DNA. The reactions were run at 3 different temperatures (48°C, 53°C, and 58°C) using 4 different DNA polymerases. HotStarTaq d-Tect shows highly specific PCR results over the whole temperature range, whereas other polymerases either failed or gave highly unspecific bands.

Increased mismatch discrimination.

[B] During annealing,methylation-specific primers also often bind to the unmethylated, converted DNA with a mismatch or several mismatches at the 3' end of the primer. [A] and [B]Taq DNA Polymerase can efficiently elongate these primers, regardless of the primer mismatch. Due to its increased ability to discriminate between mismatches, genetically engineered HotStarTaq d-Tect Polymerase recognizes a mismatch at the 3' end of the methylation-specific primer, and prevents primer extension. [C] HotStarTaq d-Tect Polymerase only elongates primers without mismatch. [D] Thus, HotStarTaq d-Tect Polymerase increases reliability in MSP PCR, and prevents false-positive amplification reactions, facilitating primer design.

Performance

Novel genetically engineered polymerases, such as HotStarTaq d-Tect included in QIAGEN’s EpiTect MSP Kit, provide high PCR specificity (see figure "Sensitivity of HotStarTaq d-Tect"). This polymerase only extends primers that perfectly match the target DNA at the 3' primer end (the starting point of primer extension), enabling analysis of single CpG sites. This is in contrast to wild-type DNA polymerases, which often ignore 3' mismatches and extend mis-primed primers, producing nonspecific, false-positive methylation results (see figure "Increased mismatch discrimination").

Principle

The EpiTect MSP Kit enables simple development of new methylation-specific PCR assays. Primer design for MSP can be difficult, due to the need for each primer to cover several CpG sites. This has prevented the analysis of a single CpG site within a CpG island. However, since HotStarTaq d-Tect Polymerase only requires a single base mismatch at the 3' end of one primer, designing primers for MSP is simplified, and sensitive analysis of the methylation status of several and even individual CpG sites can be performed (see figure "Single base discrimination").

Procedure

The EpiTect MSP Kit offers a master mix format, allowing convenient and easy reaction setup. HotStarTaq d-Tect Polymerase is provided in an inactive state, with no polymerase activity at ambient temperatures. This prevents the formation of misprimed products and primer dimers at low temperatures. HotStarTaq d-Tect Polymerase is activated by a 10-minute, 95°C incubation step, which can easily be incorporated into existing thermal cycling programs.

Standardized workflows in epigenetics

Accessing epigenetic information is of prime importance for many areas of biological and medical research — particularly oncology, but also stem cell research and developmental biology. However, the analysis of changes in DNA methylation is challenging due to the lack of standardized methods for providing reproducible data, particularly from limited sample material. With its newly introduced EpiTect solutions, QIAGEN makes available standardized, pre-analytical and analytical solutions: from DNA sample collection, stabilization and purification, to bisulfite conversion and real-time or end-point PCR methylation analysis or sequencing (see figure "Standardized workflows in epigenetics").